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More than 150 familial Alzheimer disease (FAD)-associated missense mutations in presenilins (PS1 and PS2), the catalytic subunit of the gamma-secretase complex, cause aberrant amyloid beta-peptide (Abeta) production, by increasing the relative production of the highly amyloidogenic 42-amino acid variant. The molecular mechanism behind this pathological activity is unclear, and different possibilities ranging from a gain of function to a loss of function have been discussed. gamma-Secretase, signal peptide peptidase (SPP) and SPP-like proteases (SPPLs) belong to the same family of GXGD-type intramembrane cleaving aspartyl proteases and share several functional similarities. We have introduced the FAD-associated PS1 G384A mutation, which occurs within the highly conserved GXGD motif of PS1 right next to the catalytically critical aspartate residue, into the corresponding GXGD motif of the signal peptide peptidase-like 2b (SPPL2b). Compared with wild-type SPPL2b, mutant SPPL2b slowed intramembrane proteolysis of tumor necrosis factor alpha and caused a relative increase of longer intracellular cleavage products. Because the N termini of the secreted counterparts remain unchanged, the mutation selectively affects the liberation of the intracellular processing products. In vitro experiments demonstrate that the apparent accumulation of longer intracellular cleavage products is the result of slowed sequential intramembrane cleavage. The longer cleavage products are still converted to shorter peptides, however only after prolonged incubation time. This suggests that FAD-associated PS mutation may also result in reduced intramembrane cleavage of beta-amyloid precursor protein (betaAPP). Indeed, in vitro experiments demonstrate slowed intramembrane proteolysis by gamma-secretase containing PS1 with the G384A mutation. As compared with wild-type PS1, the mutation selectively slowed Abeta40 production, whereas Abeta42 generation remained unaffected. Thus, the PS1 G384A mutation causes a selective loss of function by slowing the processing pathway leading to the benign Abeta40.

Amyloid [beta]-peptide (A[beta]) is generated by the consecutive cuts of two membrane-bound proteases. [beta]-Secretase cuts at the N terminus of the A[beta] domain, whereas [gamma]-secretase mediates the C-terminal cut. Recent evidence suggests that the presenilin (PS) proteins, PS1 and PS2, may be [gamma]-secretases. Because PSs principally exist as high molecular weight protein complexes, biologically active [gamma]-secretases likely require other cofactors such as nicastrin (Nct) for their activities. Here we show that preferentially mature Nct forms a stable complex with PSs. Furthermore, we have down-regulated Nct levels by using a highly specific and efficient RNA interference approach. Very similar to a loss of PS function, down-regulation of Nct levels leads to a massive accumulation of the C-terminal fragments of the [beta]-amyloid precursor protein. In addition, A[beta] production was markedly reduced. Strikingly, down-regulation of Nct destabilized PS and strongly lowered levels of the high molecular weight PS1 complex. Interestingly, absence of the PS1 complex in [PS1.sup.-/-] cells was associated with a strong down-regulation of the levels of mature Nct, suggesting that binding to PS is required for trafficking of Nct through the secretory pathway. Based on these findings we conclude that Nct and PS regulate each other and determine [gamma]-secretase function via complex formation.

The Alzheimer's disease (AD)-associated presenilin (PS) proteins are required for the [gamma]-secretase cleavages of the/3-amyloid precursor protein and the site 3 (S3) protease cleavage of Notch. These intramembrane cleavages release amyloid-[beta] peptide (A/3), including the pathogenic 42-aa variant (A[beta].sub.342]), as well as the [beta]-amyloid precursor protein and the Notch intracellular domains (AICD, NICD). Whereas A[beta] is generated by endoproteolysis in the middle of the transmembrane domain, AICD and NICD are generated by cleavages at analogous positions close to the cytoplasmic border of the transmembrane domain. Numerous mutations causing familial AD (FAD) that all cause increased production of A[beta]42 have been found in the PS1 gene. Here we have investigated the previously uncharacterized, very aggressive FAD mutation L166P that causes onset of AD in adolescence. Strikingly, the PS1 L166P mutation not only induces an exceptionally high increase of A[beta].sub.42] production but also impairs NICD production and Notch signaling, as well as AICD generation. Thus, FAD-associated PS mutants cannot only affect the generation of NICD, but also that of AICD. Moreover, further analysis with artificial L166 mutants revealed that the [gamma]-secretase cleavage at position 40/42 and the S3-like [gamma]-secretase cleavage at position 49 of the A[beta] domain are both differentially affected by PS1 L166 mutants. Finally, we show that PS1 L166 mutants affect the generation of NICD and AICD in a similar manner, supporting the concept that S3 protease and S3-like [gamma]-secretase cleavages are mediated by identical proteolytic activities.

Author(s): Karim S. Echtay [1]; Edith Winkler [1]; Martin Klingenberg (corresponding author) Uncoupling proteins (UCPs) are thought to be intricately controlled uncouplers [1, 2, 3] that are responsible for the [...]

Research has been conducted on the site-specific mutagenesis. The acidic side chain function in nucleotide binding and in H (super)+ and Cl (super)- transport has been investigated.

Woodward reagent K (WRK) was applied to the uncoupling protein (UCP) to prove the hypothesis that the transport inhibiting nucleotide binding to the UCP is regulated by the dissociation state of a carboxyl group in UCP with pK 4.5, and to identify the carboxyl group. Findings indicated that the pH-sensing carboxyl group controls both the nucleoside di- and triphosphate binding. Acid residue E190 found in the fourth transmembrane helix was identified as the carboxyl group.

A fast and convenient multistep purification procedure for the endogenous enzyme [gamma]-secretase, the protease complex that cleaves the Alzheimer's disease-associated [beta]-amyloid precursor protein (APP) and a number of other type I membrane proteins is reported. The developed procedure could also be used to purify [gamma]-secretase from cell lines stably overexpressing presenilin (PS) and biochemical analysis of [gamma]-secretase carrying a mutant catalytic subunit.

Based on the discovery of coenzyme Q (CoQ) as an obligatory cofactor for [H.sup.+] transport by uncoupling protein 1 (UCP1) [Echtay, K. S., Winkler, E. & Klingenberg, M. (2000) Nature (London) 408, 609-613] we show here that UCP2 and UCP3 are also highly active [H.sup.+] transporters and require CoQ and fatty acid for [H.sup.+] transport, which is inhibited by low concentrations of nucleotides. CoQ is proposed to facilitate injection of [H.sup.+] from fatty acid into UCP. Human UCP2 and 3 expressed in Escherichia coli inclusion bodies are solubilized, and by exchange of sarcosyl against digitonin, nucleotide binding as measured with 2'-O-[5-(dimethylamino)-naphthalene-1-sulfonyl]-GTP can be restored. After reconstitution into vesicles, [Cl.sup.-] but no [H.sup.+] are transported. The addition of CoQ initiates [H.sup.+] transport in conjunction with fatty acids. This increase is fully sensitive to nucleotides. The rates are as high as with reconstituted UCP1 from mitochondria. Maximum activity is at a molar ratio of 1:300 of CoQ:phospholipid. In UCP2 as in UCP1, ATP is a stronger inhibitor than ADP, but in UCP3 ADP inhibits more strongly than ATP. Thus UCP2 and UCP3 are regulated differently by nucleotides, in line with their different physiological contexts. These results confirm the regulation of UCP2 and UCP3 by the same factors CoQ, fatty acids, and nucleotides as UCP1. They supersede reports that UCP2 and UCP3 may not be [H.sup.+] transporters.

Pathogenic generation of amyloid β-peptide (Aβ) by sequential cleavage of β-amyloid precursor protein (APP) by β- and γ-secretases is widely believed to causally underlie Alzheimer disease (AD). β-Secretase initially cleaves APP thereby generating a membrane-bound APP C-terminal fragment, from which γ-secretase subsequently liberates 37-43-amino acid long Aβ species. Although the latter cleavages are intramembranous and although lipid alterations have been implicated in AD, little is known of how the γ-secretase-mediated release of the various Aβ species, in particular that of the pathogenic longer variants Aβ42 and Aβ43, is affected by the lipid environment. Using a cell-free system, we have directly and systematically investigated the activity of γ-secretase reconstituted in defined model membranes of different thicknesses. We found that bilayer thickness is a critical parameter affecting both total activity as well as cleavage specificity of γ-secretase. Whereas the generation of the pathogenic Aβ42/43 species was markedly attenuated in thick membranes, that of the major and rather benign Aβ40 species was enhanced. Moreover, the increased production of Aβ42/43 by familial AD mutants of presenilin 1, the catalytic subunit of γ-secretase, could be substantially lowered in thick membranes. Our data demonstrate an effective modulation of γ-secretase activity by membrane thickness, which may provide an approach to lower the generation of the pathogenic Aβ42/43 species. [ABSTRACT FROM AUTHOR]

The two proteases β-secretase and γ-secretase generate the amyloid β peptide and are drug targets for Alzheimer's disease. Here we tested the possibility of targeting the cellular environment of β-secretase cleavage instead of the β-secretase enzyme itself. β-Secretase has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both compounds mildly raised the membrane-proximal, endosomal pH and inhibited β-secretase cleavage at therapeutically achievable concentrations in cultured cells, in primary neurons, and in vivo in guinea pigs. This shows that an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit β-secretase. Surprisingly, bepridil and amiodarone also modulated γ-secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target β- and γ-secretase through distinct molecular mechanisms. In addition to Alzheimer's disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins. [ABSTRACT FROM AUTHOR]

?-Secretase is an intramembrane cleaving protease involved in Alzheimer's disease.?-Secretase occurs as a high molecular weight complex composed of presenilin (PS1/2), nicastrin (NCT), anterior pharynx-defective phenotype 1 and PS enhancer 2. Little is known about the cellular mechanisms of?-secretase assembly. Here we demonstrate that the cytoplasmic tail of PS1 fulfills several functions required for complex formation, retention of unincorporated PS1 and?-secretase activity. The very C-terminus interacts with the transmembrane domain of NCT and may penetrate into the membrane. Deletion of the last amino acid is sufficient to completely block?-secretase assembly and release of PS1 from the endoplasmic reticulum (ER). This suggests that unincorporated PS1 is actively retained within the ER. We identified a hydrophobic stretch of amino acids within the cytoplasmic tail of PS1 distinct from the NCT-binding site, which is required to retain unincorporated PS1 within the ER. Deletion of the retention signal results in the release of PS1 from the ER and the assembly of a nonfunctional?-secretase complex, suggesting that at least a part of the retention motif may also be required for the function of PS1. [ABSTRACT FROM AUTHOR]

Examines the role of regulated presenilin (PS1) and nicastrin (Nct) in identifying amyloid Β-peptide production via complex formation. Function of β-secretase and γ-secretase; Levels of the molecular weight of PS1 complex; Inhibition of Nct expression RNA.

Examines the pH dependence of nucleotide binding to uncoupling protein (UCP) using radioactively labeled nucleotides. Biochemical description of UCP; Identification of incorporation site; Guanosine triphosphate as the preferred nucleotide.